Figure 1.
The original Nnd-1 allele is present in the SAT Nnd-1 stock.
(A) DSC Nnd-1 stock is homozygous for the Harding-Theobald et al. [40] deletion, as the deletion 3′ primer does not yield the 290 bp PCR product. (B) SAT Nnd-1 stock contains the original Nnd1 allele, as the 290 bp PCR product is present at a low level (lane with white asterisk). (C) In a sub-sample of 9 iso-chromosomal lines isolated from the SAT Nnd-1 stock one is revealed as the original Nnd-1 chromosome (lane with white asterisk). Photographs of ethidium bromide stained agarose gels are shown. PCR included one 5′ N primer (5′ primer 1) and two 3′ N primers; one located downstream of the deletion (3′ primer 1 that yields the 670 bp product) and one inside the deletion (deletion 3′ primer that yields the 290 bp product from alleles without the Theobald-Harding et al. deletion). (D) PCR primers flanking the Theobald-Harding et al. deletion (5′ primer 1 and 3′ primer 2) yield PCR products of different sizes indicating that this deletion is present in the DSC Nnd-1 allele but not in the original Nnd-1 allele. A 307 bp product was expected with the Theobald-Harding et al. deletion (from the DSC Nnd-1 allele) and a 348 bp product was expected without this deletion (from the original Nnd-1 allele). - = no template control; M = marker DNA. (E) Schematic representation of the Theobald-Harding et al., deletion (∇), primers used determine its presence or absence in fly lines (arrow heads), and polymorphisms (↓p1 – p5) detected in the study (p1 = CAA to CAG in the eighth Glutamine codon in the opa region; p2 = CAA to CAG in the last Glutamine codon in the opa region; p3 = G to C at position 8161; p4 = a T deletion at position 8300; and p5 = a T insertion at position 8544. Coding region polymorphisms p1 and p2 do not change the amino acid sequence, confirming the earlier report by others that there is no change in the amino acid sequence of the original nd1 allele (FlyBase).
Figure 2.
The mutation in the DSE and not polymorphisms or T deletion/insertion in the 3′ UTR of the original Nnd1 allele affects mRNA expression in Drosophila S2 cells.
(A) Northern blots showing that the 3′ UTR of the Nnd1 allele and 3′ UTR of the wild type N allele produce comparable levels of GFP mRNA. Both constructs contained the actin promoter, GFP coding region, and the wild type N DSE. (B) Northern blots showing that addition of the Nnd1 DSE mutation to the Nnd1 3′ UTR affects mRNA expression in Drosophila S2 cells. Nnd1-dse lane = actin promoter +GFP coding+ Nnd1 3′ UTR + Nnd1-dse; N wt lane = actin promoter +GFP coding+ N 3′ UTR + N dse. rp49 = RNA loading control. (C) Poly(A) Tail assay (PAT) of RNA samples used in Fig. 2B showing that polyadenylation of residual Nnd1-dse mRNA is comparable to the level of polyadenylation of wild type (wt) N mRNA (lanes 3 and 4). A 10∶1 ratio of Nnd1-dse:wt total RNA was used as it provided comparable levels of N RNA as determined by northern blots (bottom panel). PAT assay was performed using a N specific (5′ primer 2, see Fig. 4D) and an oligo d(T) primer that initiates DNA synthesis all along the length of the poly(A) tail, thereby revealing the level of polyadenylation of N mRNA. Lane 2 is the product of PCR using N cDNA template (control template) and an N primer (3′ primer 3, see Fig. 4D) ending at the N mRNA cleavage site to indicate the size of the fragment without the poly(A) tail. Ethidium bromide gel images are shown. no RT = reverse transcriptase omitted for cDNA synthesis.
Figure 3.
Nnd1-dse mutation is in the GU-rich Down Stream Element (DSE) of the N consensus poly(A) site.
Important features of the poly(A) site are marked on the actual sequencing read-out of the Nnd1-dse sequence revealing the site of mutation.
Figure 4.
Nnd1-dse embryos produce higher levels of poly(A)-tailed N mRNA and Nintra.
(A) Northern blots showing comparable levels of N mRNA in Nnd1-dse and wild type embryos after 60 minutes at the restrictive temperature of 30°C. (B) PAT assay using N specific (5′ primer 2) or rp49 specific primer and an oligo d(T) primer that reveals the level of polyadenylation of N mRNA (top panel) and the control rp49 mRNA (bottom panel). The smear of fragments of heterogeneous lengths that is present only in the Nnd1-dse lane indicates that N mRNA is poly(A)-tailed to a higher level in Nnd1-dse embryos than in wild type embryos. Lane 1 is the product of PCR using N cDNA template (control template) and an N primer (3′ primer 3) ending at the N mRNA cleavage site. Ethidium bromide gel images are shown. no RT = reverse transcriptase omitted in the cDNA synthesis reaction. rp49 = rp49 PAT fragments amplified from the same samples that served as controls. (C) Unprocessed (extended) transcript assay using one primer upstream of the mRNA cleavage site (5′ primer 2) and one primer downstream of the cleavage site (3′ primer 4). Only N transcripts that bypass the consensus poly(A) site are expected to be amplified. To assess the level of total RNA in the reactions, primers located within the rp49 cDNA were used. Ethidium bromide gel images are shown. D. Schematic representation of the primers used for results presented in B and C.
Figure 5.
Nnd1-dse embryos show gain-of-N signaling molecular and developmental phenotypes.
(A) Western blots showing that Nnd1-dse embryos overproduce Nintra. Statistical analysis of values standardized to the level of hsp70 showed that Nnd1-dse embryos produce 28.8X higher Nintra and 9.2X lower full length N compared to wild type embryos (p<0.01 for both, n = 3). N = full length N protein; * = non-specific band; *1 = the dominant-negative NΔCterm fragment [22], [25]. (B) Neuronal cells are lost to varying degrees in Nnd1-dse embryos, as expected with the gain in N signaling. Embryos were probed for the Hunchback protein, a neurogenesis marker.